Phenolic acid compounds in calyx sinica as well as extraction method and application thereof

Abstract

The invention discloses a phenolic acid compound in calystegia hederacea and an extraction method and application of the phenolic acid compound, the structure of the phenolic acid compound is determined as a new compound by adopting 1H-NMR, 13C-NMR and two-dimensional nuclear magnetic spectrum analysis methods, and the phenolic acid compound has the following structure: # imgabs0. The phenolic acid compound disclosed by the invention can effectively inhibit the activity of acetylcholin esterase (AChE); the compound disclosed by the invention can be used as a lead compound for resisting acetylcholin esterase, and a material basis and a theoretical basis are provided for developing new drugs and new components.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine extraction and separation, and relates to four new phenolic acid compounds, extraction methods and uses. Background Art

[0002] Lethariella cladonioides Nyl. Krog, also known as Jinshua Ba, Hongxuecha and Luxinxuecha, is a branched lichen body of the genus Lethariella, subgenus Chlorea, family Parmaliaceae. Lethariella cladonioides Nyl. Krog only exists in East Asia, and its distribution area in China involves six provinces and regions including Shaanxi, Gansu, Qinghai, Tibet, Yunnan and Sichuan, and the distribution center is mainly in southeastern Tibet, northwestern Yunnan and western Sichuan. "Shaanxi Chinese Herbal Medicine" records that it is bitter in taste and neutral in nature, and has the functions of sedation, anti - inflammation and pain relief; it is mainly used to treat epilepsy, schizophrenia, neurasthenia, headache and dizziness.

[0003] Lethariella cladonioides Nyl. Krog mainly includes various chemical components such as depsides, phenolic acids, aromatic esters and aldehydes, steroids, polyols and volatile oils, thus showing a wide range of pharmacological activities. Modern pharmacological research shows that Lethariella cladonioides Nyl. Krog has antioxidant, lipid - lowering, anti - tumor, anti - fatigue, antibacterial, anti - radiation and other effects.

[0004] At present, most of the chemical components isolated from Lethariella cladonioides Nyl. Krog are known, and the structural novelty is low, but the extraction of phenolic acid compounds from Lethariella cladonioides Nyl. Krog, extraction methods and uses have not been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide phenolic acid compounds.

[0006] The second purpose of the present invention is to provide an extraction method for phenolic acid compounds.

[0007] The third purpose of the present invention is to provide the uses of phenolic acid compounds.

[0008] The fourth purpose of the present invention is to provide an extract of Lethariella cladonioides Nyl. Krog containing phenolic acid compounds.

[0009] The fifth purpose of the present invention is to provide the uses of the extract of Lethariella cladonioides Nyl. Krog.

[0010] The sixth purpose of the present invention is to provide a pharmaceutical composition containing phenolic acid compounds.

[0011] The seventh purpose of the present invention is to provide the uses of the above - mentioned pharmaceutical composition.

[0012] The technical solution of the present invention is outlined as follows:

[0013] Phenolic acid compounds having the structure shown in formula (I):

[0014]

[0015] The extraction method of the above phenolic acid compounds comprises the following steps:

[0016] Step 1: Using the dried herbs of Jin Si Shua as raw materials, extracting with ethanol-water, filtering, combining the filtrates, concentrating under reduced pressure and heating, concentrating until there is no alcohol smell, cooling to room temperature, and obtaining an extract for standby;

[0017] Step 2: Dissolving the extract obtained in Step 1 with pure water, extracting with petroleum ether and dichloromethane, recovering the solvent under reduced pressure, and obtaining a three-part extract;

[0018] Step 3: Separating the dichloromethane extraction layer obtained in Step 2 by silica gel column chromatography, eluting with petroleum ether, petroleum ether-ethyl acetate, ethyl acetate, and methanol in a gradient manner, and obtaining 14 fractions (Fr.d1-14) for standby;

[0019] Step 4: Further separating the fraction Fr.d9 obtained in Step 3 by silica gel column chromatography, respectively using petroleum ether-ethyl acetate for gradient elution, and obtaining 3 fractions (Fr.d91, Fr.d92, Fr.d93);

[0020] Step 5: Separating the fraction Fr.d92 obtained in Step 4 by Sephadex LH-20, detecting by thin-layer chromatography, and combining and concentrating according to the color development results for standby;

[0021] Step 6: Separating the concentrate obtained in Step 5 by reverse-phase silica gel column chromatography, eluting with methanol-water as the mobile phase, detecting by thin-layer chromatography, and combining and concentrating according to the color development results for standby;

[0022] Step 7: Separating and preparing the concentrate obtained in Step 6 by HPLC, eluting with methanol-water as the mobile phase, and preparing 5 fractions (Fr.d921-Fr.d925).

[0023] Step 8: Separating and preparing the fraction Fr.d923 obtained in Step 7 by HPLC, eluting with methanol-water as the mobile phase, and preparing Compound I-1.

[0024] Step 9: Separating and preparing the fraction Fr.d922 obtained in Step 7 by HPLC, eluting with methanol-water as the mobile phase, and preparing Compounds I-2, I-3 and I-4.

[0025] The preferred step 1 is as follows: Using the dried medicinal materials of Jin Si Shua as raw materials, adding an ethanol solution of 95% which is 8 to 16 times the mass of the raw materials, performing ultrasonic extraction 1 to 3 times, 30 minutes each time, then adding an ethanol solution of 75% which is 8 to 16 times the mass of the raw materials, performing ultrasonic extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract; or using the dried medicinal materials of Jin Si Shua as raw materials, adding an ethanol solution of 95% or 75% which is 8 to 16 times the mass of the raw materials, performing ultrasonic extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract; or using the dried medicinal materials of Jin Si Shua as raw materials, adding an ethanol solution of 95% which is 8 to 16 times the mass of the raw materials, performing heating extraction 1 to 3 times, then adding an ethanol solution of 75% which is 8 to 16 times the mass of the raw materials, performing heating extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract; or using the dried medicinal materials of Jin Si Shua as raw materials, adding an ethanol solution of 95% or 75% which is 8 to 16 times the mass of the raw materials, performing heating reflux extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract.

[0026] The application of the above phenolic acid compounds in the preparation of anti-acetylcholinesterase drugs.

[0027] The Jin Si Shua extract containing the above phenolic acid compounds.

[0028] The application of the Jin Si Shua extract in the preparation of anti-acetylcholinesterase drugs.

[0029] A pharmaceutical composition, characterized by comprising phenolic acid compounds or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers and / or excipients.

[0030] The application of the above pharmaceutical composition in the preparation of anti-acetylcholinesterase drugs.

[0031] Advantages of the present invention:

[0032] The phenolic acid compounds of the present invention can effectively inhibit the activity of acetylcholinesterase (AChE), indicating that the phenolic acid compounds of the present invention can be used as lead compounds for anti-acetylcholinesterase. Detailed implementation manners

[0033] The technical solutions of the present invention will be described below in combination with specific embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] Example 1

[0035] The extraction method of phenolic acid compounds includes the following steps:

[0036] Step 1: Using the dried whole herb (4.0 kg) of Lethariella cladonioides Nyl. Krog as raw material, cut it into pieces, and ultrasonically extract it 3 times (30 min) with 14 times the amount of 95% ethanol and 75% ethanol respectively. Filter, combine the filtrates, and recover the solvents under reduced pressure at 40°C - 45°C to obtain 340 g of extract.

[0037] Step 2: Take the above extract, dissolve it with pure water, extract it with equal amounts of petroleum ether and dichloromethane, and recover the solvents from the extract under reduced pressure to obtain the dichloromethane layer extract (66.7 g).

[0038] Step 3: After dissolving the dichloromethane layer extract, add 1.2 times the amount of silica gel to mix the sample, and perform silica gel column chromatography separation. Use petroleum ether, petroleum ether - ethyl acetate with volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, 2:1, 1:1 and ethyl acetate, methanol as eluents for gradient elution to obtain 14 fractions (Fr.d1 - Fr.d14).

[0039] Step 4: Mix the fraction Fr.d9 with 1.3 times the amount of silica gel, perform silica gel column chromatography separation, and use petroleum ether - ethyl acetate with volume ratios of 10:1, 8:1, 4:1, 2:1, 1:1 and ethyl acetate, methanol as eluents for gradient elution to obtain 3 fractions (Fr.d91, Fr.d92, Fr.d93).

[0040] Step 5: First purify Fr.d92 with Sephadex LH - 20 CH2Cl2 - MeOH (1:1, v / v), and then perform ODS column chromatography, using methanol - water with volume ratios of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and methanol as eluents for gradient elution.

[0041] Step 6: Perform HPLC separation and preparation on the above sample, use methanol - water as the mobile phase for isocratic elution, and prepare 5 fractions (Fr.d921, Fr.d922, Fr.d923, Fr.d924, Fr.d925).

[0042] Step 7: Perform HPLC separation and preparation on Fr.d924, use methanol - water with a volume ratio of 80:20 as the mobile phase for isocratic elution to obtain Compound 1.

[0043] The physical and chemical properties and constants of each compound are as follows:

[0044] Compound I-1: White powder; UV (methanol) λmax (logε) 203 (5.28), 245 (4.34), 278 (4.45) nm; IR (KBr) vmax 3442, 2915, 1683, 1440, 1207, 1139, 1022, 956 cm -1 ; High-resolution mass spectrometry (cation) m / z 303.0869 [M+H] + (Calculated molecular formula C 16 H 15 O6, 303.0869); Carbon and hydrogen NMR data are shown in Table 1.

[0045] Compound I-2: White powder; UV (methanol) λmax (logε) 205 (4.66), 270 (4.23), 304 (4.00) nm; IR (KBr) vmax 3420, 2994,, 1662, 1613, 1437, 1315, 1256, 1203, 1140, 1016, 951 cm -1 ; High-resolution mass spectrometry (cation) m / z 347.1129 [M+H] + (Calculated molecular formula C 18 H 19 O7, 347.1131); Carbon and hydrogen NMR data are shown in Table 1.

[0046] Compound I-3: White powder; (c 0.016 g / 100 mL, methanol); UV (methanol) λmax (logε) 204 (3.51), 231 (3.11), 265 (2.85), 311 (2.43) nm; IR (KBr) vmax3419, 2914, 1647, 1456, 1398, 1318, 1262, 1175, 1092, 1022, 961 cm -1 ; High-resolution mass spectrometry (cation) m / z 429.1187 [M+H] + (Calculated molecular formula C 22 H 21 O9, 429.11876); Carbon and hydrogen NMR data are shown in Table 1.

[0047] Compound I-4: White powder; (c = 0.0086 g / 100 mL, methanol); UV (methanol) λmax (logε) 204 (3.96) nm; IR (KBr) vmax 3402, 2949, 2838, 1645, 1453, 1415, 1285, 1111, 1022 cm -1 ; High-resolution mass spectrometry (cation) m / z 435.0921 [M+H]+ (Calculating the molecular formula C 20 H 19 O 11 , 435.0922); The data of carbon-hydrogen NMR spectra are shown in Table 1.

[0048] Table 1 The data of hydrogen and carbon NMR spectra of Compounds I-1 to I-4

[0049]

[0050] By means of physical and chemical constants and modern spectroscopy (MS and NMR), combined with relevant literature data, its structure was identified. Compounds I-1 to I-4 are new compounds not reported in the literature. As follows:

[0051]

[0052] Example 2

[0053] Step 1: Using the dried whole herb (10.0 kg) with a golden wire brush as the raw material, cut it into pieces, and extract it with 10 times the amount of 95% ethanol and 75% ethanol by heating under reflux for 3 times (30 min) respectively, filter, combine to obtain the filtrate, and recover the solvent under reduced pressure at 30 °C to 45 °C to obtain the extract.

[0054] Steps 2 to 9 are the same as Steps 2 to 9 in Example 1 to prepare Compounds I-1 to I-4

[0055] Example 3

[0056] Acetylcholinesterase (AChE) inhibitory activity test of phenolic acid compounds

[0057] Preparation of AChE solution: Dissolve it with 10 mL of PBS buffer to prepare a 0.05 U / ml AChE solution. Preparation of ATCI solution: Weigh 21.7 mg of ATCI precisely into a centrifuge tube, add 10 mL of PBS buffer solution to the centrifuge tube and dissolve it fully to prepare a 7.5 mmol / L solution. Store it in the dark at 4 °C for later use. Preparation of DTNB solution: Weigh 39.6 mg of DTNB precisely into a centrifuge tube, then add 10 mL of phosphate buffer solution to the centrifuge tube and dissolve it fully to prepare a 10 mmol / L solution (prepare it immediately before use). Preparation of compound solution: Weigh the compound precisely and dissolve it in DMSO, and dilute the compound to different concentrations with PBS buffer solution as needed. Similarly, prepare galantamine solution as a positive control. Add 140 μL of phosphate buffer solution, 20 μL of sample solution and 20 μL of AChE solution into a 96-well plate in sequence, oscillate to mix them evenly, and then place them in an incubator at 30 °C for 15 min. Then add 10 μL of DTNB and 10 μL of ATCI, oscillate evenly and place them in an incubator at 37 °C for 30 min. Finally, use an enzyme-labeled instrument to measure the absorbance value (OD) of each well at 412 nm. Use galantamine instead of the test sample solution for the positive control, add 20 μL of PBS instead of the sample solution for the standard group, and add 40 μL of PBS instead of the test compound solution and AChE solution for the blank group. Each group of data is parallel for 3 times, and the inhibition rate is calculated according to the following formula:

[0058] Inhibition rate (%) = (ODstandard - ODsample) / (ODstandard - ODblank) × 100%

[0059] Finally, use SPSS software to calculate the half maximal inhibitory concentration (IC 50 ) value of the test solution and the positive control drug against AChE.

[0060] Table 2 Results of the inhibitory activity of compounds against AChE

[0061]

[0062] The acetylcholinesterase inhibitory drug containing the compound or composition of the present invention can be in dosage forms suitable for oral administration or injection, etc. For example, it can be tablets, capsules, granules, injections, pills, syrups, powders, etc.

[0063] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications are also included in the protection scope of the claims of the present invention.

Claims

1. Four phenolic acid compounds extracted and isolated from golden wire brushes, characterized in that, The molecular formulas are respectively: C 16 H 14 O6, C 18 H 18 O7, C 22 H 20 O9, and their chemical names are: 3-hydroxy-5-methylphenyl-3-formyl-2,4-dihydroxy-6-methylbenzoate 4-(ethoxycarbonyl)-3-hydroxy-5-methylphenyl-2,4-dihydroxy-6-methylbenzoate 1,10-Diethoxy-1,3-dihydro-4-hydroxy-5,8-dimethyl-3,7-dioxo-7H-isobenzofuro[4,5-b][1,4]benzodioxepin-11-carbaldehyde 1,4-dihydroxy-1,3-dihydro-10-methoxy-5-methoxymethyl-8-methyl-3,7-dioxo-7H-isobenzofuro[4,5-b][1,4]benzodioxepin-11-carbaldehyde. The chemical structural formula is as follows:

2. The extraction method of the compound according to claim 1, characterized in that It includes the following steps: Step 1: Using the dried medicinal materials of golden wire brush as raw materials, adding ethanol-aqueous solution, extracting, filtering, combining the filtrates and recovering ethanol under reduced pressure, concentrating until there is no ethanol left, cooling to room temperature, and obtaining the extract for standby; Step 2: Dissolving the extract obtained in Step 1 with pure water, extracting with petroleum ether and dichloromethane, recovering the solvent under reduced pressure, and obtaining the three-part extract; Step 3: Separating the dichloromethane extraction layer obtained in Step 2 by silica gel column chromatography, eluting with petroleum ether, petroleum ether-ethyl acetate, ethyl acetate, and methanol in a gradient manner, detecting by thin-layer chromatography, combining according to the color development results, and concentrating to dryness under reduced pressure to obtain 14 fractions (Fr.d1-Fr.d14) for standby; Step 4: Separating the fraction Fr.d9 obtained in Step 3 by silica gel column chromatography again, eluting with petroleum ether-ethyl acetate, ethyl acetate, and methanol in a gradient manner respectively, obtaining several elution parts, detecting by thin-layer chromatography, combining according to the color development results, and concentrating to dryness under reduced pressure to obtain 3 fractions (Fr.d91, Fr.d92, Fr.d93); Step 5: Separating the fraction Fr.d92 obtained in Step 4 by Sephadex LH-20, detecting by thin-layer chromatography, and combining and concentrating for standby according to the color development results; Step 6: Separating the concentrate obtained in Step 5 by reverse-phase silica gel column chromatography, eluting with methanol-water as the mobile phase, detecting by thin-layer chromatography, and combining and concentrating for standby according to the color development results; Step 7: Separating and preparing the concentrate obtained in Step 6 by HPLC, eluting with methanol-water as the mobile phase, and preparing 5 fractions (Fr.d921-Fr.d925); Step 8: Separating and preparing the fraction Fr.d923 obtained in Step 7 by HPLC, eluting with methanol-water as the mobile phase, and preparing the compound I-1; Step 9: The fraction Fr.d922 obtained in Step 7 was subjected to HPLC separation and preparation, eluted with methanol-water as the mobile phase, and compounds I-2, I-3 and I-4 were prepared.

3. The method according to claim 2, wherein Step 1 is: using the dried medicinal materials with a golden wire brush as the raw material, adding an ethanol solution of 95% with a weight 8 to 16 times that of the raw material, performing ultrasonic extraction 1 to 3 times for 30 minutes each time, then adding an ethanol solution of 75% with a weight 8 to 16 times that of the raw material, performing ultrasonic extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract; or using the dried medicinal materials with a golden wire brush as the raw material, adding an ethanol solution of 95% or 75% with a weight 8 to 16 times that of the raw material, performing ultrasonic extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract; or using the dried medicinal materials with a golden wire brush as the raw material, adding an ethanol solution of 95% with a weight 8 to 16 times that of the raw material, performing heating extraction 1 to 3 times, then adding an ethanol solution of 75% with a weight 8 to 16 times that of the raw material, performing heating extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract; or using the dried medicinal materials with a golden wire brush as the raw material, adding an ethanol solution of 95% or 75% with a weight 8 to 16 times that of the raw material, performing heating reflux extraction 1 to 3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until there is no ethanol left to obtain an extract.

4. Use of the phenolic acid compound of claim 1 in the preparation of an anti-acetylcholinesterase drug.

5. A golden wire brush extract containing the phenolic acid compound of claim 1.

6. Use of the golden wire brush extract of claim 5 in the preparation of an anti-acetylcholinesterase drug.

7. A pharmaceutical composition, characterized in that Comprising the phenolic acid compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

8. Use of the pharmaceutical composition of claim 7 in the preparation of an anti-acetylcholinesterase drug.

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